Genetic selection system for identification of MicroRNA target genes

ABSTRACT

There is provided an expression cassette comprising a 3′-UTR cDNA library fragment, mammalian cells transfected with the expression cassette, and kits comprising the same. Furthermore, methods for identifying target genes for microRNAs are provided that utilize the expression cassette hereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a nonprovisional of and claims priority to U.S.Provisional Application Ser. No. 61/000,336, filed Oct. 25, 2007, whichdocument is hereby incorporated by reference to the extent permitted bylaw.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant numberW81XWH-06-1-0604 and grant number W81XWH-08-1-0658 awarded by the U.S.Army Medical Research Acquisition Activity. The government has certainrights in this invention.

BACKGROUND OF THE INVENTION

The present invention generally relates to an expression cassettecomprising a 3′-UTR cDNA library fragment, mammalian cells transfectedwith the expression cassette, and kits comprising the same. Furthermore,methods for identifying target genes for microRNAs are provided thatutilize the expression cassette hereof.

MicroRNAs are a class of naturally-occurring small non-coding RNAs thatcontrol gene expression by translational repression or mRNA degradation(1-3). They are abundantly expressed and could comprise 1-5% of animalgenes (4). Since the discovery of lin-4 and let-7 in Caenorhabditiselegans (5-7), over six thousand microRNAs have been identified in avariety of organisms, including plants, flies and animals through thegenomics and bioinformatics effort (8). Like protein-coding genes,microRNAs are transcribed as long primary transcripts (pri-microRNAs) inthe nucleus. However, distinct from protein-coding genes, they aresubsequently cleaved to produce stem loop structured precursor molecules(pre-microRNAs) of 70-100 nucleotides (nt) in length by the nuclearRNase III enzyme Drosha (9). The pre-microRNAs are then exported to thecytoplasm by exportin-5 (10) where the RNase III enzyme Dicer furtherprocesses them into mature microRNAs (˜22 nt). One strand of themicroRNA duplex is subsequently incorporated into the RNA-inducedsilencing complex (RISC) that mediates target gene expression. Althoughthe microRNA pathways leading to gene silencing are not fully understoodyet, evidence indicates that they target mRNAs for translationalrepression or mRNA cleavage (11, 12). Since microRNAs are able tosilence gene expression by binding to the 3′-untranslated region(3′-UTR) of the gene with partial sequence homology, a single microRNAusually has multiple targets (13). Thus, microRNAs could regulate alarge fraction of protein-coding genes. Indeed, as high as 30% of allgenes could be microRNA targets (11, 14). In essence, microRNAs can beconsidered to be modulators of gene regulators and they can cooperatewith transcription factors. Together, microRNAs and transcriptionfactors determine gene expression patterns in the cell (15). Therefore,the discovery of microRNAs adds a new layer of gene regulation to thecomplex gene expression network.

Given the important role of microRNA in regulating cellular pathways, ithas been found that a unique set of microRNAs (or microRNA signatures)are often associated with human cancer. Lu et al. reported a generaldownregulation of a number of microRNAs in tumors compared with normaltissues in multiple human cancers (16). Of considerable interest,microRNA expression profiles are able to successfully classify poorlydifferentiated tumors whereas mRNA profiles are highly inaccurate forthe same samples (16). MicroRNA signatures have also been reported inother types of cancers, including chronic lymphocytic leukemia (CLL)(17), lung cancer (18), pituitary adenomas (19), uterine leiomyomas (20)and adult acute myeloid leukemia (AML) (21). In lung cancer, microRNAexpression profiles correlate with survival of lung adenocarcinomas,including those classified as disease stage I; high miR-155 and lowlet-7a-2 expression correlates with poor survival (18). Hierarchicalclustering analysis of microRNA expression profiles is able todistinguish tumor from normal pancreas, pancreatitis and cell lines(22). In pituitary adenomas, 30 microRNAs are differentially expressedbetween normal pituitary and pituitary adenomas and among them, 24microRNAs can serve as a predictive signature of pituitary adenoma and29 microRNAs are able to predict pituitary adenoma histotype (19). Inhuman uterine leiomyomas, 31 of 206 microRNAs examined reveal a distinctmicroRNA expression profile associated with tumor size and race (20).More interestingly, a solid cancer microRNA signature is suggested by alarge portion of overexpressed microRNAs from a large-scale miRnomeanalysis on 540 samples, including lung, breast, stomach, prostate,colon, and pancreatic tumors (23). Together, these findings highlightthe potential of microRNA profiling in cancer diagnosis (16).

The fundamental role of microRNAs in regulating cellular pathwayssuggests that deregulation of microRNAs affects normal cell growth anddevelopment, leading to a variety of disorders including neurologicaldiseases (24) and human cancer (12, 25-28). Specific overexpression orunderexpression has been shown to correlate with particular tumor types(16, 17, 32-34) because overexpression of a particular set of microRNAscould result in down-regulation of tumor suppressor genes, whereas theirunderexpression could lead to oncogene up-regulation, suggesting thatmicroRNAs may function as either oncogenes or tumor suppressor genes(29). Since microRNAs are often located at fragile sites or inrepetitive genomic sequences of chromosomal regions (30), this mayexplain why microRNA expression deregulation occurs frequently in humancancer. For instance, 68% of investigated patients suffering from B-cellchronic lymphocytic leukemia (CLL) have been shown to have a deletionlocated at chromosome 13q14 where the miR-15 and miR-16 genes reside andare under-represented in many B-CLL patients (31).

Apparently, whether a microRNA functions as an oncogene or tumorsuppressor is largely determined by the target genes of each particularmicroRNA. For example, tumor suppressive microRNAs, such as let-7,miR-15 and miR-16, are able to suppress expression of oncogenes. let-7suppresses ras oncogene and is downregulated in lung cancer (32); miR-15and miR-16 suppress Bcl-2 anti-apoptotic gene, and they are deleted ordownregulated in leukemia (31, 33). In contrast, oncogenic microRNAs cansilence tumor suppressor genes. miR-17-5p and miR-20a control thebalance of cell death and proliferation driven by the proto-oncogenec-Myc (34) and miR-17-5p serves as an oncogene in lymphoma and lungcancer (35, 36). Similarly, a cluster consisting of miR-372 and miR-373have been shown to function as oncogenes in testicular germ cell tumorsby suppressing the p53 pathway (37). Moreover, it has been demonstratedby the present inventors and others that antisense miR-21oligonucleotide suppresses tumor cell growth which is associated withincreased apoptosis and decreased cell proliferation (38, 39) therebysuggesting that miR-21 is an oncogene. The present inventors and otherssubsequently identified the tumor suppressor gene tropomyosin 1 (TPM1)as a direct miR-21 target gene (40). Furthermore, miR-21 also plays arole in cell invasion and tumor metastasis, which is likely throughregulation of multiple miR-21 target genes, such as TPM1, programmedcell death 4 (pdcd4) and maspin (41). Of interest, certain microRNAs mayspecifically modulate only tumor metastasis. For example, miR-10bfunctions as a metastasis initiation factor and overexpression ofmiR-10b causes breast tumor invasion and metastasis, but it has noeffect on primary tumor growth (42). On the other hand, miR-335suppresses metastasis and migration through targeting of the progenitorcell transcription factor SOX4 and extracellular matrix componenttenascin C (43).

Since microRNAs regulate cellular pathways by suppression of theirspecific target genes, identification of microRNA target genes iscritical to the understanding of molecular mechanisms ofmicroRNA-mediated tumorigenesis. Computational algorithms have been amajor driving force in predicting microRNA targets (44-46). Theapproaches are mainly based on base pairing between microRNA and targetgene 3′-UTR, emphasizing the location of microRNA complementary elementsin 3′-UTR of target mRNAs, the concentration in the seed sequence (6-8bp) of continuous Watson-Crick base pairing in 5′ proximal half of themicroRNA and the phylogenetic conservation of the complementarysequences in 3′-UTRs of orthologous genes. They provide very usefulprimary sources in search for microRNA targets. However, despite thefundamentally similar approaches used for the published screens formicroRNA targets, predicted targets for a given microRNA often varyamong different methods. Presumably the approaches differ in certainimportant details, such as defining phylogenetic conservation,thermodynamic and statistical factors applied to score and rankpredicted sites. The fact that mature microRNAs are short and typicallycontain several sequence mismatches with their target transcripts hascomplicated computational target predictions. This might explain whycomputer-aided algorithms are still unable to provide a precise pictureof microRNA regulatory networks. In addition, a recent report indicatesthat perfect seed pairing is not a generally reliable predictor formiRNA-target interactions at least in some cases (47, 48), which furtherhighlights the difficulty of microRNA target predictions. Thus, they canonly serve a complementary approach and certainly need in vivoexperimental validations. Another challenge is that it is not clearwhether a microRNA can target mRNA which does not carry a putativebinding site for this microRNA. If this is the case, such a target genemay escape from these prediction methods because all of them are mainlybased on sequence homology between microRNA and mRNA. More recently,there are reports that microRNAs are able to bind to 5′-UTR or codingregions and silence or even enhance the corresponding genes. Thesefindings suggest that microRNAs are not necessarily restricted to the3′-UTR to exert their function. However, the currently predictionmethods are mainly based on the 3′-UTR. In other words, some microRNAtargets would also escape from these prediction methods.

Regarding microRNA prediction methods, currently there is no clearconsensus as to which one is most reliable. The present inventors havecompared four commonly cited microRNA target prediction programs,TargetScan4 (49), miRBase Target5(http://microrna.sanger.ac.uk/targets/v5/), PicTar (50) and miRanda(http://www.microma.org) (51). In general, miRBase Target5 and miRandatend to predict more targets than TargetScan4 or PicTar does presumablybecause the first two programs do not weigh as much on conservationsamong different species as the other two programs. Using miR-21 as anexample, miRBase Target5 and miRanda predict 1000 and 2501 targets,respectively. On the other hand, TargetScan4 and PicTar predict 186 and175 targets, respectively. However, only a small fraction of predictedtargets among these methods overlap thereby suggesting that each methodhas its own unique set of parameters. For example, some of these modelshave recently been refined to consider the presence of secondarystructures and other features of the 3′-UTR sequence surrounding thetarget site, and for the ability of complementarity at the 3′ end of thecognate miRNA to compensate for imperfect seed matching (49, 52).Nevertheless, despite these efforts, little is known about theprediction accuracy of these methods because only a very limited numberof targets have been experimentally validated. Therefore, there is aneed in the art for systematic target validation methods.

Microarray technology could be one of target validation approachesbecause it is capable of determining expression of potential microRNAtargets at the mRNA level (53, 54). However, given that a large fractionof microRNA target genes are silenced by the translation repressionmechanism, those microRNA targets may escape from the microarraydetection. Alternatively, microRNAs can be used as endogenouscytoplasmic primers to synthesize cDNA on an mRNA template (55) suchthat recovered primers would presumably be functional microRNAs.However, this is technically challenging because of limited sequencehomology between mRNA and microRNA. In addition, it could be extremelydifficulty to recover those microRNAs that can cause mRNA degradation.Alternatively, biochemical or proteomic methods have been used for thispurpose (56-61), but they could be labor intensive.

Currently, in research laboratories a common approach to validatewhether a gene is a direct microRNA target involves cloning of the3′-UTR of this gene into a reporter (e.g., luciferase), followed byreporter assays. It is further verified to be suppressed by a givenmicroRNA at the mRNA level (e.g., real-time RT-PCR) or at the proteinlevel (e.g., Western blot). Apparently, validation of multiple microRNAtargets with this approach needs a high throughput screening systembecause each microRNA will have to be individually tested against agiven UTR sequence, which requires intensive labor and costly reagents(FIG. 13 right). Therefore, the selection system described here willsave tremendous time and cost because this method allows selection ofpositive microRNA/mRNA interactions (FIG. 13 left).

The genetic selection method of the present invention represents aunique systematic validation system for microRNA targets that provides acomprehensive picture of microRNA/mRNA interactions for a given gene ora given microRNA. One of the advantages of this system is that it allowsfor the determination of microRNA/mRNA interactions whether mRNAdegradation or translation repression is involved or whether conservedmicroRNA binding sites are required. Moreover, this is a simple butpowerful selection method that does not require intensive labor orcostly instrument and reagents and it is suitable for a large number ofmicroRNAs or target genes.

The following references that are referred throughout this disclosureare hereby incorporated by reference in their entirety to the extentpermitted by law. These references merely serve to support the inventionand to provide background and context. Applicant reserves the right tochallenge the veracity of any statements therein made.

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SUMMARY OF THE INVENTION

In one of many illustrative, non-limiting aspects of the presentinvention, there is provided an expression cassette comprising a 3′-UTRcDNA library fragment, mammalian cells transfected with the expressioncassette, and kits comprising the same. Furthermore, methods foridentifying target genes for microRNAs are provided that utilize theexpression cassette hereof. The following abbreviations and terms areused throughout the specification and have the following definitions:

When introducing elements of the present invention or embodiments(s)thereof, the articles “a”, “an”, “the” and “said” are intended to meanthat there are one or more of the elements. The terms “comprising”,“including” and “having” are intended to be open and inclusive and meanthat there may be additional elements other than the listed elements.

A “bp” is an abbreviation for base pair.

A “ds” is an abbreviation for double-stranded.

A “GFP” is an abbreviation for green fluorescent protein.

An “nt” is an abbreviation for nucleotide.

A “target gene” refers to any gene suitable for regulation ofexpression, including both endogenous chromosomal genes and transgenes,as well as episomal or extrachromosomal genes, mitochondrial genes,chloroplastic genes, viral genes, bacterial genes, animal genes, plantgenes, protozoal genes and fungal genes.

A “library” as used herein refers to a collection of nucleic acidsequences that possesses a common characteristic. For example, a libraryof nucleic acids can be representative of all possible configurations ofa nucleic acid sequence over a defined length. Alternatively, a nucleicacid library may be a collection of sequences that represents aparticular subset of the possible sequence configurations of a nucleicacid of a defined length. A library may also represent all or part ofthe genetic information of a particular organism. A nucleic acid“library” is typically, but not necessarily, cloned into a vector.

The term “gene” refers to a nucleic acid (e.g., DNA) sequence thatcomprises coding sequences necessary for the production of a polypeptideor precursor or RNA (e.g., tRNA, siRNA, rRNA, etc.). The polypeptide canbe encoded by a full length coding sequence or by any portion of thecoding sequence so long as the desired activity or functional properties(e.g., enzymatic activity, ligand binding, signal transduction, etc.) ofthe full-length or fragment are retained. The term also encompasses thecoding region of a structural gene and the sequences located adjacent tothe coding region on both the 5′ and 3′ ends, such that the genecorresponds to the length of the full-length mRNA. The sequences thatare located 5′ of the coding region and which are present on the mRNAare referred to as 5′ untranslated sequences. The sequences that arelocated 3′ or downstream of the coding region and that are present onthe mRNA are referred to as 3′ untranslated sequences. The term “gene”encompasses both cDNA and genomic forms of a gene. A genomic form orclone of a gene contains the coding region, which may be interruptedwith non-coding sequences termed “introns” or “intervening regions” or“intervening sequences.” Introns are removed or “spliced out” from thenuclear or primary transcript, and are therefore absent in the messengerRNA (mRNA) transcript. The mRNA functions during translation to specifythe sequence or order of amino acids in a nascent polypeptide.

The term “expression vector” refers to both viral and non-viral vectorscomprising a nucleic acid expression cassette.

The term “expression cassette” is used to define a nucleotide sequencecontaining regulatory elements operably linked to a coding sequence thatresult in the transcription and translation of the coding sequence in acell.

A “mammalian promoter” refers to a transcriptional promoter thatfunctions in a mammalian cell that is derived from a mammalian cell, orboth.

A “mammalian minimal promoter” refers to a ‘core’ DNA sequence requiredto properly initiate transcription via RNA polymerase binding, but whichexhibits only token transcriptional activity in the absence of anyoperably linked transcriptional effector sequences.

The phrase “open reading frame” or “coding sequence” refers to anucleotide sequence that encodes a polypeptide or protein. The codingregion is bounded in eukaryotes, on the 5′ side by the nucleotidetriplet “ATG” that encodes the initiator methionine and on the 3′ sideby one of the three triplets which specify stop codons (i.e., TAA, TAG,and TGA).

“Operably linked” is defined to mean that the nucleic acids are placedin a functional relationship with another nucleic acid sequence. Forexample, a promoter or enhancer is operably linked to a coding sequenceif it affects the transcription of the sequence; or a ribosome bindingsite is operably linked to a coding sequence if it is positioned so asto facilitate translation. Generally, “operably linked” means that theDNA sequences being linked are contiguous. However, enhancers do nothave to be contiguous. Linking is accomplished by ligation at convenientrestriction sites. If such sites do not exist, the syntheticoligonucleotide adaptors or linkers are used in accord with conventionalpractice.

“Recombinant” refers to the results of methods, reagents, and laboratorymanipulations in which nucleic acids or other biological molecules areenzymatically, chemically or biologically cleaved, synthesized,combined, or otherwise manipulated ex vivo to produce desired productsin cells or other biological systems. The term “recombinant DNA” refersto a DNA molecule that is comprised of segments of DNA joined togetherby means of molecular biology techniques.

“Transfection” is the term used to describe the introduction of foreignmaterial such as foreign DNA into eukaryotic cells. It is usedinterchangeably with “transformation” and “transduction” although thelatter term, in its narrower scope refers to the process of introducingDNA into cells by viruses, which act as carriers. Thus, the cells thatundergo transfection are referred to as “transfected,” “transformed” or“transduced” cells.

The term “plasmid” as used herein, refers to an independentlyreplicating piece of DNA. It is typically circular and double-stranded.

A “reporter gene” refers to any gene the expression of which can bedetected or measured using conventional techniques known to thoseskilled in the art.

Other objects and features will be in part apparent and in part pointedout hereinafter.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

In the accompanying drawings that form a part of the specification andthat are to be read in conjunction therewith:

FIG. 1 is a schematic representation of a plasmid pSSMT1 carrying bothtTR-KRAB and tetO-Pu in accordance with one embodiment of the presentinvention;

FIG. 2 is a schematic representation of the construction of a pSSMT1library in accordance with one embodiment of the present invention;

FIG. 3A is a schematic representation of one embodiment of the selectionmethod of the present invention;

FIG. 3B is a schematic representation of another embodiment of theselection method of the present invention;

FIG. 4 is a schematic representation of primers derived from knownsequences flanking the inserts in accordance with one embodiment of thepresent invention;

FIG. 5 is a schematic representation of the selection system inaccordance with one embodiment of the present invention;

FIG. 6A is a schematic representation of validation of miR-21 targetTPM1 by the selection system in accordance with one embodiment of thepresent invention;

FIG. 6B is a schematic representation of validation of miR-21 targetTPM1 by the selection system in accordance with another embodiment ofthe present invention;

FIG. 7A is a western blot showing validation of miR-21 target TPM1 bythe selection system in accordance with one embodiment of the presentinvention;

FIG. 7B is a graphical representation of the western blot of FIG. 7A;

FIG. 8A is a representation of a programmed cell death 4 (PDCD4)/miR-21target in accordance with one embodiment of the present invention;

FIG. 8B is a representation of a maspin/miR-21 target in accordance withone embodiment of the present invention;

FIG. 8C is a graphical representation showing that PDCD4 is a directtarget for miR-21 in accordance with one embodiment of the presentinvention;

FIG. 8D is a graphical representation showing that maspin is a directtarget for miR-21 in accordance with one embodiment of the presentinvention;

FIG. 9A is a graphical representation showing suppression ofLuc-cytokeratin 8 UTR by miR-21 in accordance with one embodiment of thepresent invention;

FIG. 9B is a representation of a gel-electrophoresis analysis showingthe downregulation of the GFP protein by miR-21 when the cytokeratin 8UTR was cloned downstream of GFP in accordance with one embodiment ofthe present invention;

FIG. 10A is an immunostain that demonstrates transfected MCF-7 cellswith locked nucleic acid LNA anti-miR-21 oligo and then immunostainedwith anti-PDCD4 antibody;

FIG. 10B is an immunostain that demonstrates non-transfected MCF-7cells;

FIG. 11A is a western blot showing PDCD4 protein levels in 8 pairs ofmatched breast tumor specimens;

FIG. 11B is a graphical representation of a statistical analysis usingthe Pearson's method confirming the inverse correlation between PDCD4protein and miR-21 in the tissue samples of FIG. 11A with a correlationcoefficient of −0.824;

FIG. 12A is an immunohistochemical stain showing a negative correlationbetween miR-21 and PDCD4 in matched breast tumor specimens;

FIG. 12B is an in situ hybridization of the tumor specimens of FIG. 12A;

FIG. 13A is a schematic representation of one embodiment of theselection method of the present invention;

FIG. 13B is a schematic representation of a prior art screening method;

FIG. 14A is a schematic representation of constructs in accordance withone embodiment of the present invention;

FIG. 14B is a graphical representation of the survival rates of theconstructs of FIG. 14A in accordance with one embodiment of the presentinvention;

FIG. 15A is a table summarizing putative mir-21 targets; and

FIG. 15B is a table providing genes for target validation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is generally directed to a genetic selectionsystem capable of identifying target genes for microRNAs. In particular,there is provided herein an expression cassette including at least onerepressor, at least one 3′-UTR cDNA library fragment operably linked tothe repressor, an operator gene corresponding to the repressor which isoperably linked to a constitutive promotor, and an antibiotic resistancegene operably linked to the constitutive promotor.

As one skilled in the art will appreciate, the expression cassettehereof is expressible in and/or transforms any mammalian cells suitablefor use in the present invention. In certain embodiments, a mammaliancell can be a mammalian cell that is isolated from an animal (i.e., aprimary cell) or a mammalian cell line. Methods for cell isolation fromanimals are well known in the art. In some embodiments, a primary cellis isolated from a mouse. In other embodiments, a primary cell isisolated from a human. In still other embodiments, a mammalian cell linecan be used. Exemplary cell lines include HEK293 (human embryonickidney), HT1080 (human fibrosarcoma), NTera2D (human embryonicteratoma), HeLa (human cervical adenocarcinoma), Caco2 (human colonadenocarcinoma), HepG2 (human liver hepatocellular carcinoma), Cos-7(monkey kidney), ES-D3 (mouse embryonic stem cell), BALBC/3T3 (mousefibroblast), hES H1 (human embryonic stem cell), MCF-7 and MDA-MB-231(human breast cancer). Host cell lines are typically available from, forexample, the American Tissue Culture Collection (ATCC), any approvedBudapest treaty site or other biological depository. In still otherembodiments, a mammalian embryonic stem (ES) cell can be used, such as amouse ES cell mES-D3 or a human ES cell hES H1.

The expression cassette of the present invention may be contained in aplasmid, shuttle vector, viral vector, or the like, and the expressioncassette or plasmid hereof may include, in a 5′ to 3′ direction, atleast one repressor, at least one fusion protein, and combinationsthereof. To enable the selection method of the present invention, theexpression cassette hereof is also configured to be transfected by amicroDNA-expressing vector capable of gene suppression. SuitablemicroDNA include, but are not limited to, miR-21, miR-15, miR-16,miR-17-5p, miR-20a, miR-372, miR-373, miR-335, miR-10b, miR-30, miR-224,and let-7.

Suitable repressors include, but are not limited to, tetracyclinerepressors (tetR), Lac I, and combinations thereof. Alternatively or incombination, a fusion protein may be used. A fusion protein is derivedfrom a repressor and a repressor domain of a protein. In an illustrativeexample, a plasmid pSSMT-1 was constructed to carry tTR-KRAB which is afusion protein derived from tetracycline repressor (tTR) and a repressordomain of the human Kox1 zinc finger protein (64). This fusion proteinhas been shown to tightly control the target gene expression by bindingto the corresponding tetO (65-67). Moreover, tTR-KRAB is able toeffectively silence gene expression from tetO sequences placed more than3 kb from the transcriptional start site (65).

In certain embodiments, the expression cassette of the present inventionmay also include a 3′-untranslated region (3′-UTR) cDNA library orlibrary fragment operably linked to the repressor, the fusion protein,or a combination thereof. It will be appreciated by one skilled in theart that any 3′-UTR cDNA library or library fragment suitable for use inthe present invention may be used including tumor-specific UTRlibraries, pathway-specific UTR libraries, and genome-wide cDNAlibraries. However, the quality and complexity of the library is likelya major factor determining how many potential targets can be selectedout. Thus, one way to improve its selection efficiency would be to uselibraries from various sources because some genes are only expressedunder a certain circumstance or in a different tissue. For example, itis possible for maspin to be identified only from a normal breastlibrary but not from the tumor library. Therefore, libraries suitablefor use in the present invention may include, but not limited to, normaltissue libraries and libraries derived from breast tumors, lung tumors,stomach tumors, prostate tumors, colon tumors, pancreatic tumors,chronic lymphocytic leukemia, pituitary adenomas, uterine leiomyomas, oradult acute myeloid leukemia.

Moreover, to generate a 3′-UTR library in pSSMT-1, commerciallyavailable cDNA libraries may be used that were made from various tumorspecimens or normal tissues or even cell lines. In particular, thoselibraries having cDNA inserts that can be easily released from thevectors by EcoRI and NotI and which are compatible with the cloningsites in pSSMT-1 are particularly useful. Most libraries are made usingthe oligo-dT as a primer during reverse transcription and shouldtherefore carry the 3′-UTRs. In the illustrative examples discussedhereinbelow, cDNA libraries from tumor specimens were the primary UTRsources. However, given that a large set of genes involved in basiccellular processes can avoid microRNA regulation due to short 3′-UTRs,microRNA binding sites could be specifically depleted (71) or alteredthrough alternative splicing. Furthermore, tumor cells tend to expressdifferent gene patterns than normal tissues. Therefore, it is alsowithin the scope of the present invention to generate UTR libraries fromnormal cDNAs of corresponding tissues.

In an illustrative example, a 3′-UTR library was constructed bymigrating a breast tumor cDNA library clone (Invitrogen) into thepSSMT-1 plasmid resulting in pSSMT-1-Lib (FIG. 2). One skilled in theart will also appreciate that, the 3′-UTR library hereof does notnecessarily need to be a true 3′-UTR library because some clones carrycomplete gene coding sequences and it is believed that that an entirecoding region plus the 3′-UTR carrying a microRNA target binding site isstill able to respond to regulation by the microRNA (57). Furthermore, alibrary of this type permits a determination as to whether any sequencesin addition to the 3′-UTR can be responsible for regulation by thetarget microRNA.

The expression cassette may also include an operator gene correspondingto the repressor. As an illustrative example, if tetracycline repressor(tetR) is being used then the corresponding operator gene, tetracyclineoperator (tetO) would be used. Suitable operator genes include, but arenot limited to, tetO, LacO, and combinations thereof.

In certain embodiments, the operator gene may be operably linked to apromotor. In one embodiment, the transcriptional effector sequence is amammalian promoter. In addition, the transcriptional effector can alsoinclude additional promoter sequences and/or transcriptional regulators,such as enhancer and silencers or combinations thereof. Thesetranscriptional effector sequences can include portions known to bind tocellular components which regulate the transcription of any operablylinked coding sequence. For example, an enhancer or silencer sequencecan include sequences that bind known cellular components, such astranscriptional regulatory proteins. The transcriptional effectorsequence can be selected from any suitable nucleic acid, such as genomicDNA, plasmid DNA, viral DNA, mRNA or cDNA, or any suitable organism(e.g., a virus, bacterium, yeast, fungus, plant, insect or mammal). Itis within the skill of the art to select appropriate transcriptionaleffector sequences based upon the transcription and/or translationsystem being utilized. Any individual regulatory sequence can bearranged within the transcriptional effector element in a wild-typearrangement (as present in the native genomic order), or in anartificial arrangement. For example, a modified enhancer or promotersequence may include repeating units of a regulatory sequence so thattranscriptional activity from the vector is modified by these changes.

In certain embodiments of the present invention, the promoters areconstitutive promoters. Constitutive promoters can be selected, e.g.,from Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter,cytomegalovirus immediate early gene (CMV) promoter, simian virus 40early (SV40E) promoter, elongation factor 1 alpha promoter (EF1a),cytoplasmic beta-actin promoter, adenovirus major late promoter, and thephosphoglycerol kinase (PGK) promoter. In one embodiment, a constitutivepromoter is a CMV promoter. In another embodiment, a constitutivepromoter is an SV40E promoter.

The expression cassette hereof may further include anantibiotic-resistant gene which may, in turn, be operably linked to thepromotor. Any antibiotic-resistant gene suitable for use in the presentinvention may be used including, but not limited to, puromycin,hygromycin, neomycin, zeocin, ampicillin, kanamycin, tetracycline,chloramphenicol, and combinations thereof.

In certain aspects, the present invention is also directed to a simpleand efficient technique for identification of physiologic targets formicroRNA. One of many advantages of this method is that it allows foridentification of those targets that carry no conserved microRNA bindingsites. Finally, this technique can be easily applied to identify targetsfor other microRNAs.

In particular, the genetic selection system of the present inventionincludes a method for identifying a protein as a target for a microRNA.This method includes a first step of introducing a plasmid into hostcells wherein the plasmid includes an antibiotic-resistant gene undertranscriptal regulation of an operator gene and a 3′-UTR cDNA libraryfragment under transcriptional regulation of a repressor genecorresponding to the operator gene. Next, a microRNA is introduced intothe host cells which are then grown in the presence of an antibioticcorresponding to the antibiotic-resistant gene. The cells containing themicroDNA and the 3′-UTR cDNA library fragment that are bound to eachother can then express the antibiotic-resistant gene. The protein canthen be identified based on the 3′-UTR cDNA fragment from the host cellsthat grows in the presence of the antibiotic.

In an illustrative example of the method of the present invention, theplasmid pSSMT-1 described hereinabove was constructed to carry tetO-Puwhich is an element that codes for puromycin gene(puromycin-N-acetyl-transferase) under tet operator (tetO). Thisantibiotic-resistant gene is able to confer resistance to puromycin whenno repressor (e.g., tetR) is bound on the tetO site. Thus, for example,when the pSSMT-1-Lib is introduced into 293T cells (chosen for hightransfection efficiency and a low level of miR-21 expression), thetransfected cells are expected to die in the presence of puromycinbecause, like pSSMT-1, the puromycin gene in pSSMT-1-Lib is repressed bytTR-KRAB (FIG. 3). However, when a miR-21 expressing vector isco-transfected into these cells, the cells with a cDNA clone carrying amiR-21 recognition site in pSSMT-1-Lib are expected to survive and formcolonies in the presence of puromycin (FIG. 3). This is because miR-21is capable of suppressing expression of tTR-KRAB by interacting with apotential miR-21 site. In contrast, no colony is formed for the vastmajority of clones which do not carry a potential miR-21 recognitionsite, just like those of un-transfected cells.

In another embodiment of the method of the present invention, microRNAsare selected from a pre-microRNA collection against a specific targetgene. One benefit of using a pre-microRNA collection is that microRNAsmay be identifiable even though those microRNAs were not in thepredicted list. For example, the following 12 genes were chosen fortarget validation (Table 2) because these genes: (1) play an importantrole in tumorigenesis or tumor resistance to chemotherapy/hormonetherapy; (2) have been previously shown to be often aberrantly expressedin tumor specimens; and (3) are likely microRNA targets.

In this embodiment, the pre-microRNA collection is introduced into hostcells by infection. After infection, each pSSMT1-xxx-UTR is introducedinto these cells. Selection may be performed, for example, in thepresence of 1.5 μg/ml puromycin. A slightly higher concentration thannormal may be used to reduce the background from the vector control.Once survival colonies are visible, they are transferred to 24-wellplates and expanded. These cells are then used as a source forextraction of genomic DNA. PCR is then carried out using the primersflanking the pre-microRNA to recover microRNA sequences. Those microRNAsare candidates that potentially target this specific gene. Thus, foreach target gene, two selections are possible—one for vector control andanother for a pre-microRNA clone.

To confirm that the recovered microRNAs are truly responsible forpuromycin resistance in this embodiment of the selection system, each ofthe recovered microRNA clones are individually introduced in order totest against a single microRNA instead of the pre-microRNA collection.Once each microRNA clone is verified by puromycin resistance, it is thendetermined whether such a microRNA clone is able to silence theendogenous gene expression by a suitable analysis technique such asWestern blot. It is also determined whether the microDNA clone it is adirect target for this microRNA or which region of the 3′-UTR sequenceis responsible for microRNA regulation. Finally, it is determinedwhether an antisense oligo against a specific microRNA will have anopposite effect on the validated targets.

In contrast to target genes that have a small number of potentialmicroRNAs, a microRNA usually has a large number of potential targets.Many microRNAs can have over a thousand of potential targets based onprediction methods. In order to determine how many of the targets arespecifically regulated by a particular microRNA, a third embodiment ofthe method of the present invention is provided wherein the selectionprocedure is reversed (i.e., target genes are selected for against aspecific microRNA). In this method, cDNAs carrying the 3′-UTR are firstcloned into a selection plasmid to generate a 3′-UTR library. It is thendetermined how many target genes can be selected out from this 3′-UTRlibrary by overexpression of a specific microRNA. Using this methodallows for further validation of predicted targets as well asidentification of new microRNA targets.

Moreover, using this method allows for the use of different types of UTRlibraries for selection purposes in accordance with the methods of thepresent invention. In a tumor-specific UTR library, a UTR libraryspecific to certain type of cancer, such as breast cancer, can begenerated. These tumor specific primers can be cloned into pSSMT1 andused in the selection against a given microRNA. In a pathway specificUTR library, 3′UTR sequences are cloned into a lentivector downstream ofthe repressor open reading frame to generate miR-Select 3′UTR libraries.For example, initial collections may include 654 human kinase and ˜200phosphatase mRNA 3′UTRs, as the products of these genes are known toplay key roles in cellular signaling, and disruption of these regulatorynetworks can lead to cancer.

In a genome-wide cDNA library, the selection plasmid ideally would carryonly 3′-UTR sequences because microRNAs are believed to interactspecifically with targets at the 3′-UTR. However, there is a technicalchallenge to separate the coding region from the 3′-UTR sequence duringconstruction of a UTR library, i.e., a pool of cDNAs, because there isno experimental sequence border between the coding region and 3′-UTR.Thus, each 3′-UTR sequence must be individually cloned to construct sucha library which could be cumbersome. Therefore, using the method of thepresent invention, it was theorized that cDNA carrying the3′-UTR plusthe coding region sequence is still able to be efficiently silenced bymicroRNAs. For comparison, tTR-KRAB was fused to the entire p27 codingregion plus the 3′-UTR (pSSMT1-p27-UTR-1), to the part of p27 codingregion plus the 3′-UTR (pSSMT1-p27-UTR-2), and to the 3′-UTR alone(pSSMT1-p27-UTR-3) (FIG. 14A), respectively. After transfection of 293Tcells with these constructs along with miR-221 expression vector andselection in the presence of puromycin (1.0 μg/ml) for 5 days,remarkable number of viable cells was recovered for all three constructs(FIG. 14B). In contrast, very few viable cells were recovered from thevector control. Importantly, there was no significant difference in cellsurvival among three p27-UTR-containing constructs (FIG. 14B).Therefore, these results suggest that it is not necessary to generate a“pure” 3′-UTR library for microRNA suppression. In other words, it isfeasible to use a 3′-UTR containing cDNA library as source to save timeand costs for reagents. Additional benefits of this UTR containinglibrary including enabling a determination as to whether microRNAs caninteract with coding regions or 5′-UTR to silence gene expression.

To generate a 3′-UTR library in pSSMT-1, commercially available cDNAlibraries may be used. For example, previously purchased several cDNAlibraries made from various tumor specimens or normal tissues or evencell lines from Invitrogen may be used. In particular, their librariescan be made in pSPORT, and cDNA inserts can be easily released from thevectors by EcoRI and NotI, which are compatible with the cloning sitesin pSSMT-1. Finally, all of the libraries may be made using the oligo-dTas a primer during reverse transcription, meaning that they all shouldcarry the 3′-UTRs. In this embodiment of the present invention, cDNAlibraries from tumor specimens are used as the primary UTR sources.However, given that a large set of genes involved in basic cellularprocesses can avoid microRNA regulation due to short 3′-UTRs, microRNAbinding sites could be specifically depleted (71) or altered throughalternative splicing. Furthermore, tumor cells tend to express differentgene patterns than normal tissues. Therefore, one embodiment of thepresent invention also the generation of UTR libraries from normal cDNAsof corresponding tissues. In this embodiment, the cDNA library is firstdigested with EcoRI and NotI and most of inserts ranging from 0.5 to 3.0kb can be isolated after gel separation. The pooled cDNA fragments canthen be ligated to the EcoRI and NotI-digested pSSMT-1. Aftertransformation, colonies formed on LB plates can be pooled and plasmidDNA can be isolated.

In certain embodiments, the present invention also contemplates a kitfor for identifying a protein as a target for a microRNA or forselecting microRNAs from a pre-microRNA collection against a specifictarget gene. The kit may include, but is not limited to, an expressioncassette comprising at least one repressor, at least one 3′-UTR cDNAlibrary fragment operably linked to the repressor, an operator genecorresponding to the repressor which is operably linked to aconstitutive promotor, and an antibiotic resistance gene operably linkedto the constitutive promotor.

EXAMPLES

The following non-limiting examples are provided to further illustratethe present invention.

Example 1 Construction of pSSMT-1

Referring now to FIG. 1, a pGL3 control vector (Promega) was used as abackbone to construct pSSMT-1. pGL3 vector control was digested withNotI, followed by filling with Klenow and self-ligation to eliminate theNotI site. The purpose of this step was to introduce a new NotI sitedownstream of tTR-KRAB to facilitate ligation of cDNA clones from thebreast tumor library (in pCMV-SPORTS from Invitrogen) at a later stage.The modified vector was then digested with Hind3 and Kpn1 to remove the240 bp SV40 promoter and insert the self-annealed adaptor sequencespGL3-Adaptor-5 (5′-CTTGGGATTTGAATAGGAA CCTGCAGGT) and pGL3-Adaptor-3(5′-AGCTACCTGCAGGTTCCTATTCAAATC CCAAGGTAC) through which a Sbf1 site(underlined) was introduced to accommodate the tTR-KRAB fragmentcarrying Kpn1 at one end and Sfb1 on the other end. To introduce the tetoperator element (tetO), tetO was amplified using primers TRE-Bgl2-5.2(AGGCGTATCACGAGGCCCTTTCGAGATCTAGTTTACCACTCCCTATCAGT, where Bgl2 wasunderlined) and TRE-Spe1-3.1 (TTACTAGTGCGGAGGCTGGAT, where Spe1 wasunderlined) from pTRE (Clontech). This tetO element that ended with Bgl2and Spe1, along with CMV promoter-Pu (1.2 kb) derived from pFIV-puro H1(System Biosciences) which ended with Spe1 and SalI was ligated to themodified vector at BamHI and SalI sites by a three way ligation,resulting in pGL3 control-tetO-Pu. Thereafter, the PCR-amplifiedtTR-KRAB fragment was cloned into Sfb1 and Kpn1 sites of pGL3control-tetO-Pu. During PCR amplification, EcoRI and NotI sites wereintroduced. The resultant plasmid was named pSSMT-1 which stands forselection system for microRNA targets.

Example 2 Construction of pSSMT-1-Lib

A 3′-UTR library was constructed by migrating a breast tumor cDNAlibrary clones (Invitrogen) into pSSMT-1, resulting in pSSMT-1-Lib (FIG.2). Although this is not a true 3′-UTR library because some clones carrycomplete gene coding sequences, a previous study suggests that an entirecoding region plus the 3′-UTR carrying a miR-21 binding site is able torespond to regulation by miR-21 (Zhu et al, J Biol Chem 2007;282:14328-14336). Furthermore, this library allows for determination ofwhether any sequences in addition to the 3′-UTR are responsible forregulation by miR-21.

Referring now to FIG. 2, a commercially available cDNA library expectedto contain fragments to carry protein-coding sequences, was subclonedinto pSSMT-1 to generate pSSMT-1-Lib. In particular, a breast tumor cDNAlibrary made in pCMV-SPORTS (Invitrogen) was digested with EcoRI andNotI. The cDNA library was digested with EcoRI and NotI and most of theinserts ranging from 0.5 to 3.0 kb were isolated after gel separation.The pooled cDNA fragments were then ligated to the EcoRI andNotI-digested pSSMT-1. After transformation, colonies formed on LBplates were pooled and plasmid DNA was isolated. After separation in anagarose gel, fragments ranging from 0.5 to 3 kb were isolated, purifiedand finally ligated to pSSMT-1 at EcoRI and NotI, resulting inpSSMT-1-Lib. Over 40,000 colonies were obtained through this procedureand the quality of this library was determined by isolating randomlypicked colonies and restriction digestion. Over 90% of colonies carry aninsert with size ranging from 0.5 kb to 2.5 kb.

Example 3 Enforced Expression of miR-21

PCR was then used to amplify the pre-miR-21 contained in DNA fragmentsfrom MCF-10A genomic DNA as described previously (Zhu et al, J Biol Chem2007; 282:14328-14336), and the expression of mature miR-21 wasconfirmed by TaqMan real-time PCR.

Example 4 Transfection

Plasmid DNA was introduced into cells by the calcium phosphate method asdescribed previously (Mo et al., J Biol Chem 2000; 275:41107-41113).Briefly, cells were seeded in 10 cm dishes 2 h before transfection. Thetransfection efficiency was monitored by sparking a 1/10 EGFP vector. Inmost cases, the transfection rate was about 75%. One day aftertransfection, the cells were split from one to two dishes. Once cellswere attached, puromycin was added at 1.0-1.5 μg/ml. To suppress miR-21expression, a locked nucleic acid (LNA) anti-miR-21 oligo was used. Tomonitor the localization of the oligo, it was labeled with FAM, a greenfluorescent dye. The transfection of LNA-anti-miR-21 was carried outusing RNAfectin (Applied Biological Materials, British Columbia, Calif.)per the manufacturer's protocol.

When pSSMT-1-Lib is introduced into 293T cells, the transfected cellsare expected to die in the presence of puromycin because like pSSMT-1,the puromycin gene in pSSMT-1-Lib is repressed by tTR-KRAB (FIG. 3).293T cells were chosen because of their high transfection efficiency andlow level of miR-21 expression (not shown). However, when the miR-21expressing vector is co-transfected into these cells, the cells with acDNA clone carrying a miR-21 recognition site in pSSMT-1-Lib areexpected to survive and form colonies in the presence of puromycin (FIG.3). This is because miR-21 is capable of suppressing expression oftTR-KRAB by interacting with a potential miR-21 site. In contrast, nocolonies are formed for the vast majority of clones which do not carry apotential miR-21 recognition site, similarly to un-transfected cells.

Example 5 Selection

After transfection, the cells were grown at 1-1.5 μg/ml puromycin. Newmedium with fresh puromycin was changed every other day. Two weekslater, when colonies were formed, they were transferred to 24-wellplates for further growth. To determine whether the surviving coloniescarried a potential miR-21 target sequence from the library, genomic DNAwas extracted from these cells and PCR was carried out to amplifypotential sequences using primers Krab-Lib-5.2(5′-TTCAGAGACTGCATTTGAAATC) and Krab-Lib-3.2 (5′-TGCCAAGCTACCTGCAGGTTG)derived from known sequences from the vector (FIG. 4). The PCR productswere re-cloned into pSSMT-1 to determine whether they still conferredresistance to puromycin. At this point, each of the potential clones wastested individually. The positive clones were selected and re-tested byluciferase assays by subcloning them into the pGL3-control vector.Finally, the clones, which tested positive both by puromycin resistancetests and luciferase assays, were sequenced following the selectionprocedure shown in FIG. 5.

Example 6 Suppression

To determine whether any sequence downstream of tTR-KRAB can suppresstTR-KRAB expression such that it confers resistance to puromycin,tropomyosin 1 (TPM1) 3′-UTR which carries a known miR-21 binding sitewas cloned into pSSMT-1 (FIG. 6) since it was shown to be functional andrespond to miR-21 regulation (Zhu et al, J Biol Chem 2007;282:14328-14336). Firstly, a Western blot was performed to determinewhether tTR-KRAB is suppressed by TPM1-UTR. As shown in FIG. 7A, a 38kDa band corresponding to tTR-KRAB fusion protein was detected in cellstransfected with pSSMT-1, but the level of this protein was reduced inthe cells transfected with pSSMT-1-TPM1-UTR, likely due to theendogenous miR-21. Overexpression of miR-21 further reduced the level oftTR-KRAB. Consistent with this result, it was found that the cellstransfected with pSSMT-1-TPM1-UTR plus miR-21 were more resistant topuromycin than those transfected with pSSMT-1-TPM1-UTR plus vectorcontrol (FIG. 7B). In contrast, the cells transfected with either pGL3control-tetO-Pu or pSSMT-1 were very sensitive to puromycin. Therefore,miR-21 specifically inhibited the protein level of tTR-KRAB and madecells more resistant to puromycin, demonstrating the feasibility of thissystem.

Example 7 Isolation

Through the selection procedures as described in FIG. 5, a total of 14putative miR-21 targets were isolated as shown in Table 1. Two of themwere programmed cell death 4 (PDCD4) and maspin proteins, which havepreviously been implicated in tumorigenesis and metastasis (Cmarik etal., Proc Natl Acad Sci USA 1999; 96:14037-14042) or carcinogenesis (Lauet al., Cancer Res 2007; 67:2107-2113). Furthermore, both PDCD4 andmaspin carry a predicted miR-21 binding side (FIG. 8) based on two miRNAtarget predicting programs, “program miRBase target”(http://microrna.sanger.ac.uk) or Targetscan(http://www.targetscan.org/). The luciferase reporter carrying the PDCD43′-UTR revealed about 60% reduction of luciferase activity by miR-21compared to the vector control (FIG. 8); deletion of the putative miR-21binding site abolished the effect (data not shown). To further determinethe regulation by miR-21, we cloned the PDCD4 3′-UTR into a modified GFPvector as described in Zhu et al, J Biol Chem 2007; 282:14328-14336. Inaddition, we made a similar finding for maspin (FIGS. 8 C and D).

On the other hand, although cytokeratin 8 caries no conserved miR-21binding site, a two-dimensional in gel differentiation (2-DIGE) analysisindicated that this gene was upregulated by anti-miR-21 oligonucleotide(not shown), also suggesting cytokeratin 8 as a miR-21 target. Thus, the3′-UTR of cytokeratin 8 was cloned into the pGL3 control vector. Theluciferase activity from Luc-cytokeratin 8 UTR was specificallysuppressed by miR-21 (FIG. 9A). Similarly, downregulation of the GFPprotein by miR-21 was detected when this cytokeratin 8 UTR was cloneddownstream of GFP (FIG. 9B).

To better characterize the effect of miR-21 on PDCD4 expression, MCF-7cells were transfected with locked nucleic acid LNA anti-miR-21 oligoand then immunostained with anti-PDCD4 antibody. Since the anti-miR-21was labeled with FAM, it was easy to detect the transfection. Asexpected, anti-miR-21 was predominantly localized to stress bodies(puncture like structures) (FIG. 10B). In addition, the transfectedcells expressed higher levels of PDCD4 than the un-transfected ones(FIG. 10B). In contrast, scrambled oligos did have not any effect onPDCD4 expression (FIG. 10A).

Example 8 Western Blot

To determine the clinical significance of miR-21 target genes, PDCD4protein levels in 8 pairs of matched breast tumor specimens wereexamined by Western blotting. As expected, lower levels of PDCD4 weredetected in tumors in all cases (FIG. 11A). To determine whether thereis any correlation between PDCD4 and miR-21, miR-21 expression was alsomeasured in these samples by TaqMan real-time PCR. The findingsindicated that all tumors revealed higher levels of miR-21 expression.Statistical analysis using the Pearson's method confirmed the inversecorrelation between PDCD4 protein and miR-21 in these tissue samples,with a correlation coefficient of −0.824 (FIG. 11B). Protein wasextracted as described previously (Zhu et al, J Biol Chem 2007;282:14328-14336) and the concentration was determined by Protein assayskit (Bio-Rad). Protein separation and immunoblot were carried outaccording to standard methods.

Example 9 Immunofluoresence Microscopy

Finally, this inverse relationship was examined at the cellular levelsby immounohsitochemistry (IHC) and in situ hybridization (ISH). As shownin FIG. 12, both PDCD4 and maspin were highly expressed in normal breasttissue (N), but lowly expressed in tumor tissue (T). In contrast, miR-21level was low in normal tissue, but high in breast tumor tissues.

Together, these results suggest that PDCD4, maspin and cytokeratin 8 arephysiologic targets for miR-21, demonstrating the feasibility of thisselection system for miRNA targets. Given the importance of miR-21 incancer, identification of these targets provides new insight intomolecular mechanisms of miR-21-mediated gene expression andtumorigenesis. Thus, as an oncogenic microRNA, miR-21 may exert itsfunction through suppression of tumor suppressor genes like PDCD4 ormaspin and special cytoskeletal proteins like cytokeratin 8, in additionto the previously identified TPM1 (Zhu et al, J Biol Chem 2007;282:14328-14336).

Immunofluoresence staining was used to determine PDCD expression inanti-miR-21 transfected cells as previously described (Wu et al., MolCancer Ther 2007; 6:1823-1830). Briefly, MCF-7 were transfected withscrambled and were fixed with 3% paraformaldehyde. Primary antibodyagainst PDCD (Rockland) was used to detect the PDCD signal, followed bya secondary antibody conjugated with Alexa Fluor 560.

Paraffin-embedded tissue was pretreated at 65° C. for 2 h, followed bydeparaffinization using standard procedures. Antigen retrieval wascarried out in antigen retrieval solution (10 mM Tris, 1 mM EDTA, pH9.0)before applying the primary Ubc9 antibody. Thereafter, the slides wereincubated for 2 h at room temperature followed by extensive washes withPBST and further incubated for 1 h at room temperature with thesecondary antibody conjugated with horse radish peroxidase (HRP). HRPactivity was detected using Histostain Plus kit (Invitrogen) accordingto the manufacturer's instruction. Finally, sections were counterstainedwith hematoxylin and mounted.

Having described the invention in detail, those skilled in the art willappreciate that modifications may be made of the invention withoutdeparting from the spirit and scope thereof. Therefore, it is notintended that the scope of the invention be limited to the specificembodiments described. Rather, it is intended that the appended claimsand their equivalents determine the scope of the invention.

We claim:
 1. A nucleic acid expression cassette expressible in mammaliancells, wherein the expression cassette comprises the following elementsin a 5′ to 3′ direction: a) a promoter; b) a repressor operably linkedto the promoter; c) a 3′-UTR cDNA library fragment including mRNA of the3′-UTR cDNA library sequence operably linked to the repressor; d) anoperator gene corresponding to the repressor, which is operably linkedto a constitutive promoter, and e) an antibiotic-resistant elementoperably linked to the constitutive promoter, wherein the mRNA encodingthe repressor comprises a fusion mRNA that also includes the mRNA of the3′-UTR cDNA library fragment.
 2. The expression cassette of claim 1,wherein the repressor is selected from the group consisting oftetracycline repressor (tetR), LacI, and combinations thereof.
 3. Theexpression cassette of claim 2, wherein the repressor is tetR.
 4. Theexpression cassette of claim 1, wherein the 3′-UTR cDNA library isselected from the group consisting of breast tumor cDNA libraries,normal cDNA libraries, other tumor cDNA libraries, and combinationsthereof.
 5. The expression cassette of claim 1, wherein theantibiotic-resistant element is selected from the group consisting ofpuromycin, hygromycin, neomycin, zeocin, ampicillin, kanamycin,tetracycline, chloramphenicol, and combinations thereof.
 6. Theexpression cassette of claim 1, wherein the constitutive promoter isselected from the group consisting of retroviral Rous sarcoma virus(RSV) long terminal repeat (LTR) promoter, cytomegalovirus immediateearly gene (CMV) promoter, elongation factor 1 alpha promoter (EF1a),simian virus early (SV40) promoter, cytoplasmic beta-actin promoter,adenovirus major late promoter, and phosphoglycerol kinase (PGK)promoter.
 7. The expression cassette of claim 6, wherein theconstitutive promoter is CMV promoter.
 8. The expression cassette ofclaim 1, wherein the expression cassette is contained in a plasmid,shuttle vector, or viral vector.
 9. A nucleic acid expression cassetteexpressible in mammalian cells, wherein the expression cassettecomprises the following elements in a 5′ to 3′ direction: a) a promoter;b) a fusion gene of tetracycline repressor and Krab gene operably linkedto the promoter; c) a 3′-UTR cDNA library fragment including mRNA of the3′-UTR cDNA library sequence operably linked to the fusion gene; d) atetracycline operator gene operably linked to a constitutive promoter,and e) an antibiotic-resistant element operably linked to theconstitutive promoter, wherein the mRNA encoding the repressor comprisesa fusion mRNA that also includes the mRNA of the 3′-UTR cDNA libraryfragment.
 10. The expression cassette of claim 9, wherein the 3′-UTRcDNA library is selected from the group consisting of breast tumor cDNAlibraries, normal cDNA libraries, other tumor cDNA libraries, andcombinations thereof.
 11. The expression cassette of claim 9, whereinthe antibiotic-resistant element is selected from the group consistingof puromycin, hygromycin, neomycin, zeocin, ampicillin, kanamycin,tetracycline, chloramphenicol, and combinations thereof.
 12. Theexpression cassette of claim 9, wherein the constitutive promoter isselected from the group consisting of retroviral Rous sarcoma virus(RSV) long terminal repeat (LTR) promoter, cytomegalovirus immediateearly gene (CMV) promoter, simian virus early (SV40) promoter,cytoplasmic beta-actin promoter, adenovirus major late promoter, andphosphoglycerol kinase (PGK) promoter.
 13. The expression cassette ofclaim 12, wherein the constitutive promoter is CMV promoter.
 14. Amammalian cell that is transformed with the expression cassette ofclaim
 1. 15. The mammalian cell of claim 14, wherein the mammalian cellis selected from the group consisting of HEK293, HT1080, NTERA-2D, HeLa,Caco2, HepG2, BALBC/3T3, MCF-7 and MDA-MB-231, and Cos-7.
 16. A methodfor identifying a protein as a target for a microRNA, the methodcomprising: a) introducing into host cells a plasmid comprising apromoter, an antibiotic-resistant element under transcriptionalregulation of an operator gene and a 3′-UTR cDNA library fragmentincluding mRNA of the 3′-UTR cDNA library sequence under transcriptionalregulation of a repressor corresponding to the operator gene; b)introducing into the host cells the microRNA; c) growing the host cellsin the presence of the antibiotic, wherein the cells which contain themicroRNA and the 3′-UTR cDNA library fragment that bind to each othercan express the antibiotic-resistant element; d) identifying the proteinbased on the 3-UTR cDNA fragment from the host cells that can grow inthe presence of the antibiotic, wherein the mRNA encoding the repressorcomprises a fusion mRNA that also includes the mRNA of the 3′-UTR cDNAlibrary fragment.
 17. The method of claim 16, wherein the repressor isselected from the group consisting of tetracycline repressor (tetR)gene, LacI, and combinations thereof.
 18. The method of claim 17,wherein the repressor is tetracycline repressor (tetR) gene.
 19. Themethod of claim 16, wherein the microRNA is selected from the groupconsisting of miR-21, miR-15, miR-16, let-7, miR-17-5p, miR-20a,miR-372, miR-373, miR-335, miR-10b, miR-30, and miR-224.
 20. The methodof claim 19, wherein the microRNA is selected from the group consistingof miR-21, miR-15, miR-16, let-7, miR-17-5p, miR-20a, miR-372, miR-373,miR-335, miR-10b, miR-30, and miR-224.
 21. The method of claim 16,wherein the host cells are selected from HEK293, HT1080, NTERA-2D, HeLa,Caco2, HepG2, BALBC/3T3, MCF-7 and MDA-MB-231, and Cos-7.
 22. The methodof claim 16, wherein the 3′-UTR cDNA library is selected from the groupconsisting of breast tumor cDNA libraries, normal cDNA libraries, othertumor cDNA libraries, and combinations thereof.
 23. The method of claim16, wherein the antibiotic-resistant element is an element that codesfor genes selected from the group consisting of puromycin, hygromycin,neomycin, zeocin, ampicillin, kanamycin, tetracycline, andchloramphenicol.
 24. The method of claim 16, wherein the step ofidentifying the protein is selected from the group consisting of PCR,Western blotting, immunohistochemistry and immunofluorescencemicroscopy.
 25. A method for identifying a protein as a target for amicroRNA, the method comprising: a) introducing into host cells aplasmid comprising a promoter, an antibiotic-resistant element undertranscriptional regulation of tetracycline operator (tetO) gene and a3′-UTR cDNA library fragment including mRNA of the 3′-UTR cDNA librarysequence under transcriptional regulation of tetracycline repressor(tetR) gene; b) introducing into the host cells the microRNA; c) growingthe host cells in the presence of the antibiotic, wherein the cellswhich contain the microRNA and the 3′-UTR cDNA library fragment thatbind to each other can express the antibiotic-resistant element; d)identifying the protein based on the 3-UTR cDNA fragment from the hostcells that can grow in the presence of the antibiotic, wherein the mRNAencoding the repressor comprises a fusion mRNA that also includes themRNA of the 3′-UTR cDNA library fragment.
 26. The method of claim 25,wherein the microRNA is selected from the group consisting of miR-21,miR-15, miR-16, let-7, miR-17-5p, miR-20a, miR-372, miR-373, miR-335,miR-10b, miR-30, and miR-224.
 27. The method of claim 26, wherein themicroRNA is selected from the group consisting of miR-21, miR-15,miR-16, let-7, miR-17-5p, miR-20a, miR-372, miR-373, miR-335, miR-10b,miR-30, and miR-224.
 28. The method of claim 25, wherein the host cellsare selected from HEK293, HT1080, NTERA-2D, HeLa, Caco2, HepG2,BALBC/3T3, MCF-7 and MDA-MB-231, and Cos-7.
 29. The method of claim 25,wherein the 3′-UTR cDNA library is selected from the group consisting ofbreast tumor cDNA libraries, normal cDNA libraries, other tumor cDNAlibraries, and combinations thereof.
 30. The method of claim 25, whereinthe antibiotic-resistant element is an element that codes for a geneselected from the group consisting of puromycin, hygromycin, neomycin,zeocin, ampicillin, kanamycin, tetracycline, and chloramphenicol. 31.The method of claim 25, wherein the step of identifying the protein isselected from the group consisting of PCR, Western blotting,immunohistochemistry, and immunofluorescence microscopy.
 32. A kitcomprising the expression cassette of claim
 1. 33. The kit of claim 32,wherein the repressor is tetR.
 34. The kit of claim 32, wherein the3′-UTR cDNA library is selected from the group consisting of breasttumor cDNA libraries, normal cDNA libraries, other tumor cDNA libraries,and combinations thereof.
 35. The kit of claim 32, wherein theantibiotic-resistant element is selected from the group consisting ofpuromycin, hygromycin, neomycin, zeocin, ampicillin, kanamycin,tetracycline, and chloramphenicol.